2.8 Angiogenesis (tube formation) assay in vitro
The in vitro angiogenic activity of HUVECs was determined by Matrigel tube formation assay. After the experimental period described above, HUVECs were stained with a cell-permeable dye, calcein (Cat#354216, Corning incorporated, USA), for 30 min and transferred to 24-well plates precoated with 150 μL/well growth factor-reduced Matrigel (Cat#354234, Corning incorporated, USA) and then incubated at 37°C in a cell culture incubator. After 12 h incubation, capillary-like tube formation was observed with a computer-assisted microscope (EVOS, Thermo Fisher Scientific, USA). Tube formation was defined as a tube-like structure exhibiting a length four times its width. The tube lengths in duplicate wells were counted and averaged using ImageJ software.